Homologous Recombination

MBGE 210 Fundamentals of Molecular Biology - Lecture 7: Homologous Recombination

• Lecture Date: March 10, 2025
• Instructor: Eda YILDIRIM, Ph.D., Assistant Professor, MBGE
• Instructor Contact: [email protected], Office: SCI 217
• Course Material: Lewins GENES XII, 12th edition and Molecular biology of the cell (Alberts) (online)

Cell Division: Mitosis

• DOI: 10.3390/cells11040704
• Flemming, W. Zellsubstanz, Kern und Zelltheilung; Von Verlag, F.C.W., Ed.; Vogel: Leipzig, Germany, 1882.

Genetic Recombination

• The alteration of genes present in an individual genome and the timing/level of expression is caused by DNA rearrangements.
• Genetic variation allows organisms to evolve in response to a changing environment.
• Genetic recombination is the set of mechanisms that causes DNA rearrangements.
• General and site-specific recombination are the two broad classes of recombination.

Homologous Recombination

• Important in both meiosis and mitosis.
• Generating diversity and chromosome segregation occur in meiosis
• Repairing DNA damage and stalled replication forks occur in mitosis

Site-Specific vs Somatic Recombination

• Site-specific recombination requires specific DNA sequences.
• Somatic recombination takes place in nongerm cells, not during meiosis, and is often linked to immune system recombination.
• Experimental use is an adaptation of recombination systems.

Homologous Recombination in Mitosis: Homologous End Joining

• Every proliferating cell needs a recombination reaction.
• General recombination mechanisms repair DNA replication errors by using the homologous end-joining reaction.
• Replication forks that have run into a break in the parental DNA template are restarted by homologous end-joining reaction, an interplay between replication and recombination.

Homologous Recombination in Meiosis

• General recombination enables accurate chromosome segregation in meiosis in fungi, plants, and animals.
• Exchange of bits of genetic information to create new DNA sequence combinations is caused by the crossing-over.
• Gene mixing's evolutionary benefit is wide-spread in single-celled and multicellular organisms through general recombination.

Homologous Recombination in Meiosis

• Crossing over requires chromosome synapsis to form chiasmata.
• Stages of meiosis related to molecular events at the DNA level.
• Synaptonemal complex: morphological structure of synapsed chromosomes.

Homologous Recombination in Meiosis - Key Definitions

• Sister Chromatid: One of two identical copies of a replicated chromosome, remaining linked at the centromere. Separates during anaphase in mitosis or anaphase II in meiosis.
• Bivalent: Structure containing all four chromatids (two representing each homolog) at the start of meiosis.

General Recombination: Base-Pairing Interactions

• Found in meiosis, following characteristics:
• Two homologous DNA molecules originally from different chromosomes undergo double helix breaks and joins to reform two intact double helices.
• Exchange site, where red double helix joins a green one, happens anywhere in homologous nucleotide sequences of participating DNA molecules.

General Recombination: Heteroduplex Joint

• A strand of one DNA molecule base-pairs to a strand of the second DNA molecule, forming a heteroduplex joint.
• The heteroduplex joint links the two double helices.
• Heteroduplex region can span thousands of base pairs.
• No nucleotide sequences are altered at the exchange site; cleavage and rejoining are so precise that not a single nucleotide is lost or gained.

General Recombination: Novel Sequence Creation

• General recombination creates novel DNA molecules, despite its precision.
• The heteroduplex joint is able to tolerate a small number of mismatched base pairs.
• Crossing over generally involves DNA molecules that are not exactly the same on either side of the joint.
• Newly generated recombinant DNA molecules (recombinant chromosomes)

General Recombination: Sequence Similarity

• An exchange reaction happens when two DNA double helices contain an extensive region of sequence similarity.
• Formation of a long heteroduplex joint involves pairing a strand from one double helix with a complementary strand from the other.

General Recombination: DNA Synapsis and Base-Pairing

• DNA synapsis is the process in which base pairs form between complementary strands from the two DNA molecules, allowing for sequence recognition.
• Base-pairing extends to guide the recombination process, so that matching DNA sequences allow the process to occur.

Double-Strand Breaks (DSB)

• DSBR model is relevant for mitotic and meiotic homologous recombination, initiated by creating a DSB in a single DNA duplex.
• Exonuclease action in 5' end resection generates 3' single-stranded ends to invade the other (donor) duplex.

Double-Strand Breaks (DSB) Initiate Recombination During Meiosis

• A single strand from one duplex displaces its counterpart in the other duplex via single-strand invasion, a branched structure called a D-loop is created.
• Strand exchange generates a stretch of heteroduplex DNA from each parent.

Double-Strand Breaks (DSB)

• Capture by annealing of the second double-strand break generates a recombinant joint molecule.
• The original DNA duplexes are connected via heteroduplex DNA and two Holliday junctions in a recombinant joint molecule.
• Resolution: the joint molecule separates into two molecules by nicking connecting strands.
• Recombinants form according to which strands nicked during resolution.

Gene Conversion

• Donor's genetic material unidirectionally transfers to the acceptor sequence.
• Transfer of genetic data occurs from intact homologous sequences with double-strand breaks (DSBs).
• Location can include sister chromatids, homologous chromosomes, and homologous sequences on same or different chromosomes.
• Occurs in mitosis and especially meiosis.
• Mediated by synthesis-dependent strand-annealing (SDSA) or double Holliday junction (HJ) dissolution.

Gene Conversion: Types and Demarcation

• Non-allelic (interlocus) gene conversion in trans occurs between paralogous sequences (boxes), where they reside on sister chromatids or on homologous chromosomes.
• Non-allelic gene-conversion events in cis occur between non-allelic gene copies on same chromosome; virtually indistinguishable from type a.
• Interallelic gene-conversion events happen between alleles located on homologous chromosomes.

Synthesis-Dependent Strand-Annealing (SDSA) Model

• SDSA is relevant for mitotic recombination because it produces gene conversions from double-strand breaks without associated crossovers.

Single-Strand Annealing (SSA) Mechanism

• SSA occurs at direct repeats between double-strand breaks.
• Resection of the double-strand break ends results in 3'-single-stranded tails.
• Complementarity between repeats allows annealing of single strands.
• The segment between direct repeats is removed following the completion of SSA.
• Human diseases from loss of sequence include insulin dependent diabetes, Fabry disease, alpha thalassemia.

Break-Induced Replication (BIR)

• BIR repairs double-strand breaks at other repeat sequences.
• Fragile sites: Sequences with repeat sequences (transposable elements) prone to DSB formation.
• Prone to breakage during DNA replication.
• BIR begins with one-ended double-strand break.
• BIR causes translocations in repeated sequences.

Break-Induced Replication: Mechanisms

• Strand invasion into homologous sequences forms a D-loop by a single-strand tail with a 3'-OH end.
• Synthesis results in a single strand region later converted into duplex DNA.
• A replication fork is formed, moving in direction to end of the template sequence.
• Synthesis of new DNA on both molecules is Resulted by resolution of the Holliday junction
• Holliday junction branch migrates to result in DNA on broken strand only. SDSA.
• Final products are achieved after resolution.

Homologous Recombination Pathways

• SDSA: DNA invasion into homologous DNA happens.Followed by synthesis, dissociation from template and annealing to the other broken DNA end.
• dHJ pathway: Following invasion/synthesis the second broken DSB end anneals to the D-loop, forming a dHJ intermediate.
• BIR: Invasion of one DSB end proceeds by the repair of DNA synthesis. Allows long distances in absence of another homologous broken end.